Adult skin consists of an epidermis generated principally by keratinocytes [1] and a complex dermis, populated by fibroblastic cells of mesenchymal origin [2] interspersed with vasculature, hair follicles, and other accessory structures. Within the dermis are histologically distinct regions: the papillary dermal layer just below the epidermal basement membrane, and the reticular dermal layer extending deeper to the hypodermal areas containing muscle and fat. There is no formal lineage map for dermal fibroblasts. Although types of dermal cells in culture have been described by elaborate morphological and biochemical criteria [3] the lineage progression from a mesenchymal dermal progenitor cell to reticular, papillary and follicular dermal cells is not understood.
Among the dermal fibroblasts appear to be a restricted population of mesenchymal stem cells (MSCs) [4] first identified as pluripotent, adherent cells of the bone marrow stroma [5]. MSCs in the adult are cells capable of giving use to a variety of mesenchymal phenotypes, including bone, cartilage, muscle, tendon, ligament, adipocytes, connective tissues, and dermis. Fleming et al. [4]) at Case Western Reserve University, have demonstrated that a small sub-population of the fibroblastic cells residing in the dermis react with monoclonal antibody SH2, a reagent that specifically labels MSCs in bone marrow [6]. The MSCs are clustered near vasculature, hair follicles, and adjacent to the epidermal basement membrane, although the significance of this pattern of localization has not yet been determined. Interestingly, Fleming and coworkers [4] also suggest that the number of MSCs in dermis, based on SH2 reactivity, decreases with the age of the patient to undetectable levels after age 20. This observation may contribute to our understanding of skin aging and its potential for self-renewal.
Relatively little is known about the embryologic development of human skin, although Holbrook and coworkers [2,7]have described a sequence of layer appearance with focus on the vasculature and appendage elements. Markers for extracellular matrix molecules, cell surface proteins, and growth factors have been used to help characterize the layers of skin during development, but the lack of model systems for in vitro differentiation has hampered progress in the field. Skin organ cultures [7] or embryonic side spheroids are the best system reported to study embryonic s development. These cultures consist of patches of full-thickness side introduced into suspension culture, where they round up to form spherical bodies. These cultures have epidermis on the outside and dermis on the inside, and they progress through a relatively normal sequence of developmental events. Because of the geometry of these bodies, it is difficult to use the spheroids to establish unequivocally the lineage progression of dermal cells.
In recent years, living skin equivalents [1] have been established, and the technology for reconstituting the epidermal layer from autologous or allogeneic keratinocytes has become highly advanced. The feeder layers for these keratinocyte cultures are generally fibroblasts in a formulation suitable for co-culture. While these skin equivalents can function in grafts, the fibroblastic feeder layers are not the same as a true, multi-layered dermis.
The principal needs for skin repair and regeneration methods and products are severe burns and skin ulcers. While there is substantial demand for improved therapy, there are no completely satisfactory products either on the market or in clinical development. The scope of the need in the United States is estimated as follows:
Partial thickness burns constitute those where the burn involves the surface layer of skin (epidermal layer) and into, but not through the underlying dermal layer. Most of these injuries are treated without graft or other tissue replacement. This conservative approach is effective because keratinocytes (the primary cell type of the epidermis layer) required to repair the epidermal layer are present in the dermis, particularly in the tissue surrounding hair follicles and sebaceous glands.
Patients with more extensive burns generally can not be adequately treated through spontaneous healing because (1) these burns frequently destroy the underlying dermal layer which provides the source of keratinocytes, (2) healing for full thickness wounds must occur from the margins, which is a long process that exposes the patient to a high risk of infection through unprotected tissue and (3) spontaneous healing will lead to serious scar-ring and skin contraction that is both cosmetically unattractive and may be physically restrictive due to loss of flexibility and range of motion.
Severe burns are a primary application for mesenchymal stem cell therapy because they represent a serious medical threat, they result In a high cost of treatment, and they require a long recovery period. Serious burns are frequently referred to hospitals with specialized burn units or for serious cases, the major burn centers.
Decubitus (pressure) ulcers are one of the continuing problems associated with treatment in nursing homes and hospitals dealing with bed-ridden patients. They occur due to localized pressure that restricts blood circulation to the skin. The ulcers may be quite large in area and penetrate to the full thickness of the dermal layer. They are difficult to heal and require substantial nursing resources.
Venous ulcers result from poor circulation, particularly in the legs, associated with aging. Age-deteriorated veins can lose the xe2x80x9cvalvexe2x80x9d function which keeps blood moving towards the heart. When this occurs, there is pooling of blood in the extremities and ineffective removal of toxins. This results in deterioration of the skin cells fed by the affected blood vessels.
Diabetic ulcers occur through an analogous process. Diabetes causes deterioration of the arteries through accumulation of advanced glycosylation end products (excess sugar that binds to proteins) and possibly sorbitol. As these arteries deteriorate they are unable to supply skin cells adequately, leading to cell death. As in the case of venous stasis, there is an underlying circulatory defect that requires correcting to gain fill healing. However, even if the defect is corrected, the s ulcers may persist unless properly treated.
A skin replacement is the ideal product for treating dermal ulcers. Repeated applications may be needed because the underlying defects are frequently not curable and the ulcers recur in 20-50% of cases,
Surgical wounds associated with the excision of skin cancers represent another major application for mesenchymal stem cell skin regeneration. Surgical wounds are frequently deep and cosmetically disfiguring. Treatment to accelerate healing and minimize scarring (there is a disproportionate incidence of skin cancers lesions on the face and neck) represents a significant need.
There are limited numbers of skin replacement products currently on the market and none of those in development appear likely to fully meet clinical needs, especially for full- and partial-thickness products. The measures of clinical success in skin replacement include (1) the ability to treat a wide range of dermal injuries; (2) the ability to replace or regenerate both the epidermis and the dermis skin layers: (3) a high degree of xe2x80x9ctakexe2x80x9d or acceptance and growth by the underlying tissue; (4) shortening of the natural healing process; and (5) minimal scarring.
There is little evidence that current products shorten the time to healing. Therefore, the potential exists to substantially expand the market with a therapeutic approach that both improves clinical outcomes and accelerates recovery.
True multilayer skin equivalents have not been previously possible as existing skin equivalents use a xe2x80x9csurrogate matrixxe2x80x9d for keratinocytes, such as processed allograft tissue or autograft skin, and all such surrogates lack a complete tissue morphogenic capacity.
Further, the very serious negative which compromises the attractiveness of dermal replacement using autograft from harvested skin is the need to create a substantial surgical injury that requires a long recovery period, exposes the patient to increased risk of infection through further disruption of the dermal barrier and is exceedingly painful.
The use of mesenchymal stem cells (MSCs) enables, for the first time, the development of an autologous dermal regeneration product. The skin repair products of the invention provide a true, morphogenic. multilayer, skin equivalent involving autologous MSC-derived dermoblasts and cultured human keratinocvtes thus comprising an autologous or autologous and allogeneic cell combination product. They provide full thickness dermal regeneration, producing accelerated healing and reduced scarring in a non-allergenic format.
In one aspect the invention provides a multilayer skin equivalent having (i) a scaffold layer incorporated with dermis forming cells, and (ii) a keratinocyte layer. The dermis-forming cells are preferably autologous and can be human mesenchymal stem cells, dermal fibroblasts (e.g., papillary or reticular dermal fibroblasts) or mixtures thereof. The scaffold is preferably type I collagen alone or type I and type III collagen in combination. It is also preferred that the dermis-forming cells be derived from the individual to be treated with the multilayer skin equivalent. Preferably, the keratinocytes are also autologous.
In another aspect, the invention provides a multilayer dermal equivalent comprising at least one dermis-forming layer selected from the group consisting of (i) a layer of at least one skin-associated extracellular matrix component containing papillary dermal fibroblasts; and (ii) a second layer of at least one skin-associated extracellular matrix component containing reticular dermal fibroblasts. Preferred embodiments of this aspect include those where the scaffold or dermis-forming layer is a multilayer selected from the group consisting of (a) a layer containing isolated papillary dermis-forming cells and a layer containing isolated reticular dermis-forming cells; (b) a layer containing isolated papillary dermis-forming cells and a layer containing isolated, culture expanded mesenchymal stem cells; and (c) a layer containing isolated reticular dermis-forming cells and a layer containing isolated, culture expanded mesenchymal stem cells. The papillary and reticular fibroblasts are preferably from the same individual, and this is preferably the individual to whom the multilayer dermal equivalent is to be administered.
In embodiments which comprise a layer of skin-associated extracellular matrix components containing papillary dermal fibroblasts in laminar relationship with a layer containing isolated mesenchymal stem cells, the product is positioned at-site on the recipient with the layer containing the mesenchymal stem cells in contact with the underlying tissue. In embodiments which comprise a layer of skin-associated extracellular matrix components containing reticular dermal fibroblasts in laminar, relationship with a layer containing isolated mesenchymal stem cells the product is positioned at-site on the recipient with the layer containing the reticular fibroblasts in contact with the underlying tissue.
In yet another aspect the invention provides a multilayer skin equivalent having (i) a scaffold layer comprising a first layer of at least one skin-associated extracellular matrix component containing papillary dermal fibroblasts and, in laminar relationship therewith, a second layer of at least one skin-associated extracellular matrix component containing reticular dermal fibroblasts; and (ii) a keratinocyte layer. The particular preferred embodiments of the dermis-forming layer are the same as those described above.
In a further aspect the invention provides an injectable composition comprising an injectable composition comprising dermis forming cells and at least one skin-associated extracellular matrix component in a pharmaceutically acceptable injectable carrier. The dermis forming cells can be isolated, culture-expanded mesenchymal stem cells, dermal fibroblasts and combinations thereof. They are preferably human and most preferably autologous. The dermal fibroblasts can be papillary dermal fibroblasts, reticular dermal fibroblasts or combinations thereof. The composition can further include keratinocytes.
Permutations of those aspects and embodiments which include the presence of MSCs with keratinocytes and/or committed or differentiated dermoblasts are those in which the various cell types can be combined ex vivo during the manufacture of the product or one or more of such cell types can be administered to the recipient of the dermal or skin equivalent product either by adding such cells to the product while in position on an area of skin to be repaired or, alternatively, can be administered in vivo systemically or locally.
With the decline in the presence of hMSCs, comes a significant decrease in of the ability of skin to regenerate the dermal layer. Our objective is to re-establish the ability of the patient to regenerate skin by providing a scaffold rich in culture expanded autologous hMSCs in contact with the wound bed. Our approach provides the ability to reproduce the normal multi-layered architecture of the skin and the normal physiology of skin turnover. The presence of an abundance of dermal progenitor cells will eliminate the need for subsequent regrafts and since the material will be entirely cells of non-foreign origin, there will be no risk of rejection or immune response.
The hMSC cell therapy of the present invention provides the first in vivo skin progenitor product for wound care. and thus is the first true dermal regeneration product. Human MSC dermal progenitors can be injected directly into the wound or ulcer site, formed into dermal skin equivalents in a scaffold or combined with keratinocytes to create the first functional multi-layer skin equivalents.
Unlike the use of non-immunologic fibroblasts or cadaveric tissue, autologous hMSCs would form new dermis under the control of local bioactive factors (or added bioactive factors) without harvesting patient autografts or rejection from allograft tissue. Under these circumstances, autologous hMSC products replicate the therapeutic success of skin autografts, accelerate healing, reduce the need to harvest patient dermis, and substantially reduce hospitalization time and cost.
The skin and dermal regeneration and equivalent products of the invention include skin-associated extracellular matrix components, such as collagen (preferably type I or type I in combination with type III), modified collagen, and/or elastin, ICAMs, NCAM, laminin, fibronectin, proteoglycans (HSPG, CSPG), tenascin, heparin binding growth factors, E-cadherin and/or fibrillin, in combination with isolated, culture-expanded mesenchymal stem cells (hMSCs). These mesenchymal stem cells are the naturally occurring progenitors which give rise to multiple structural and connective tissues, including normal dermis. Unlike skin equivalents produced from collagen substrates alone or produced with a layer of non-specific fibroblasts, the dermal regeneration product of the invention has significantly more dermal regeneration potential to reconstitute the patient""s own dermis which has degenerated through burns, ulcerations, or interrupted through acute injury or surgery. The ability to reconstitute normal dermis is due to the inclusion of purified autologous dermal progenitor cells in the multilayer skin equivalent or other product configuration where regenerating a dermal component would be clinically beneficial.
The skin and dermal regeneration and equivalent products of the invention can further include a bioactive factor which enhances proliferation, commitment or differentiation of mesenchymal stem cells into dermal components, either in vitro or in vivo. Also, the products of the invention can further include at least one pharmaceutical agent which promotes adhesion or angiogenesis of the dermal skin equivalent.
In order to obtain mesenchymal stem cells, it is necessary to isolate rare pluripotent mesenchymal stem cells from other cells in the bone marrow or other MSC source. Typically, a 10-20 cc aspirate is harvested from a patient which yields 1,000-5,000 hMSCs. Approximately 1-5 million culture-expanded autologous hMSCs are then returned in the form of a skin equivalent, which is applied as a skin patch or wound cover. The marrow or isolated mesenchymal stem cells can be autologous, allogeneic or from xenogeneic sources, and can be embryonic or from post-natal sources. The use of autologous cells is preferred. Bone marrow cells may be obtained from iliac crest, femora, tibiae, spine, rib or other medullary spaces. Other sources of human mesenchymal stem cells include embryonic yolk sac, placenta, umbilical cord, periosteum, fetal and adolescent skin, other soft tissues and blood.
The skin equivalents of the invention are indicated for use in regenerating dermis which has been lost through burn, ulceration, abrasion, laceration injury, or surgical wound. They are also suitable for treating patients who present with partial to full thickness burns, various dermal-involved ulcerations, and for regenerating tissue during plastic or reconstructive surgery. These skin equivalents contain autologous hMSCs, are dermagenic and, as such, regenerate dermis directly at the graft site where they are able to differentiate into one or more of the dermis-forming papillary, papillary follicular and reticular dermal cells. This process is known as Regenerative Dermal Tissue Therapy.
The direct dermagenic activity of hMSCs is superior to harvesting skin autografts or other dermal collagen scaffolds because hMSCs are able to recapitulate the original morphogenic (tissue-forming) events involved in dermal development. Harvested autografts or collagen substrates are not able to recruit sufficient newly formed dermal cells, which have differentiated from mesenchymal progenitor cells (hMSCs), from surrounding tissue or to accomplish this task in a suitable time period.